Women with one inherited mutation in the BRCA1 gene have a substantially increased predisposition to breast and ovarian cancer. However, focused study of the impact of specific BRCA1 mutations and, importantly, of heterozygosity at the BRCA1 locus, on the biology of breast epithelial cells and on breast carcinogenesis has been hampered by the lack of suitable human cell line models. To address this shortcoming, we propose to engineer novel human breast-derived, non-malignant cell lines using triplex-helix-based gene editing technology to introduce precisely defined genotypes at the BRCA1 locus. These genotypes will consist of wild-type, heterozygous, or homozygous mutations at the BRCA1 gene for two of the common deleterious alleles found in human familial cancers (185delAG and 5382insC). These unique matched sets of BRCA1 wild-type, heterozygous, and homozygous mutant cell lines will enable physiologically relevant comparisons between otherwise isogenic, breast-derived cells with defined genotypes for BRCA1. We will characterize these cells for key aspects of BRCA1 function, including regulation of transcription and of DNA damage responses and DNA repair. PUBLIC HEALTH RELEVANCE: Inherited mutations in the BRCA1 gene are a major factor in breast carcinogenesis, but focused study of the impact of specific BRCA1 mutations has been hampered by the lack of suitable human cell lines. To address this shortcoming, we propose to engineer novel human breast-derived, non-malignant cell lines using a novel gene editing technology to introduce precisely defined mutations in the BRCA1 gene in human breast- derived cells. These unique matched sets of cells will enable important and physiologically relevant comparisons among breast-derived cells with defined genotypes for BRCA1, and they will be made readily available to the scientific community to facilitate a wide range of carcinogenesis and cancer biology studies.